CN103421079A - Modification method of polyethyleneglycol of protein - Google Patents
Modification method of polyethyleneglycol of protein Download PDFInfo
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Abstract
The invention relates to modification method of polyethyleneglycol of protein, which takes hydrophobic chromatography chromatographic column as solid phase reaction carrier. The method comprises the following steps: as for the protein with strong hydrophobicity, absorbing the protein on the hydrophobic chromatography chromatographic column under a high-salt state, taking a polyethyleneglycol modification agent as a mobile phase, and taking salt ion for gradient elution after the completion reaction to obtain polyethyleneglycol modified protein; as for the protein with hydrophobicity, absorbing the polyethyleneglycol modification agent in the hydrophobic chromatography chromatographic column under the high salt sate, taking protein solution as the mobile phase, and taking salt ion for gradient elution after the completion reaction to obtain polyethyleneglycol modified protein. The PEG modified protein prepared by the invention has high retention rate of activity and better simplicity, and the single modified rate reaches more than 40 percent, and the retention rate of activity reaches more than 80 percent. The method provides a new process for the realization of PEG modification of the protein.
Description
Technical field
The invention belongs to the protein modification field, specifically, relate to the pegylation method of protein.
Background technology
Along with the development of genetic engineering technique, the clinical application of protein, polypeptide drug more and more comes into one's own.Yet protein, polypeptide drug ubiquity such as poor stability, Half-life in vivo is short and exist the defects such as immunogenicity seriously to restrict its application and development.Polyoxyethylene glycol (PEG) can make that the protein drug solubility behavior improves, stability strengthens, immunogenicity elimination or reduction, body-internal-circulation Increased Plasma Half-life, pharmacokinetics optimization, therefore in biotechnology and bio-pharmaceuticals exploitation, has important using value and application prospect widely.At present, PEG has been applied to the modification of multiple protein medicine, and the multiple proteins medicines such as the Asparaginase of modifying as PEG, Interferon, rabbit are ratified to be applied to clinical by food and drug administration (FDA) successively.
Yet, in the PEG modification, non-specific modification and the modification of multidigit point can bring the protein medicaments homogeneity poor, the problems such as reduced activity and purification difficult, formed that to take liquid-phase condition at present be development and the application limitation that basic PEG modifies.Although the modification condition is optimized, in actual modification reaction process, there are many by products to produce.In addition, the alkylation PEG modified outcome under liquid-phase condition often need to carry out the multistep purification step could remove impurity, as reductive agent, unreacted PEG and multidigit point modified outcome etc.Therefore, urgently develop a species specificity stronger and possess the single site PEG modifying method of controllability, and then simplify the preparation technology of corresponding modified protein when improving the pharmaceutical protein biological function.
Along with PEG is modified to going deep into of research, the solid phase modification technique progressively grown up has in recent years demonstrated with liquid phase and has modified the comparatively outstanding advantage of comparing, mainly concentrate on modification efficiency higher, modified specificity is stronger, purification technique is simple, and modify by solid phase the albumen obtained and retained better biologic activity, the PEG that this is pharmaceutical protein modifies provides new approaches.Solid phase PEG modification be generally first by protein adsorption to solid substrate, then the PEG of usining modifies preparation and flows through in the chromatographic column cocycle as moving phase, the albumen be adsorbed on post is realized to PEG modifies, after reaction to be finished finishes, the albumen of PEGization can adopt the salts solution wash-out obtain modified mixture or directly from unmodified protein and other, modify separation and purification acquisition purpose modified outcome by product.Theoretically, the solid phase modifying method can reduce the degree of freedom of intermolecular collision and protein surface group, thereby reduces the generation of PEG modification reaction byproduct in process thing, reduces the subsequent purification step.At present known solid phase modification process mainly concentrates on that to take ion exchange chromatography chromatogram and affinity chromatography chromatogram be solid phase adsorption material (reaction carriers).But find by a large amount of early-stage Study, existing ion-exchange or affinity chromatography solid phase are modified can not be applicable to all functional proteins, with regard to ion exchange chromatography chromatogram solid phase modification technique, its possible reason is to have covered the site that PEG modifies and cause the PEG modification reaction not carry out due to the electrostatic interaction between adhesion protein and ion solid phase; The affinity chromatography chromatogram requires target protein to produce specific binding with the thin layer chromatography post, and this bonding force to albumen and affinity chromatographic column has proposed higher requirement, makes range of application greatly limited.
Summary of the invention
The present invention is intended to make up the deficiency of existing liquid phase and solid phase PEG modification process, and a kind of novel protein solid polycondensation ethylene glycol modification process that the hydrophobic chromatography chromatographic column is reaction carriers of take is provided.
In order to realize the object of the invention, the pegylation method of a kind of protein of the present invention, it is to take the pegylation method that the hydrophobic chromatography chromatographic column is the protein of solid state reaction carrier.
The strong albumen (as N,O-Diacetylmuramidase, Chymetin etc.) for hydrophobicity, under high salt state by protein adsorption on the hydrophobic chromatography chromatographic column, take polyethyleneglycol modified dose as moving phase, reacted rear employing salt ion gradient elution (or low salt buffer wash-out) and obtained polyethyleneglycol modified albumen.
For the albumen a little less than hydrophobicity (as recombinant acid fibroblast growth factor rhaFGF, recombination basic fibroblast growth factor rhbFGF etc.), under high salt state, polyethyleneglycol modified dose is adsorbed on the hydrophobic chromatography chromatographic column, take protein solution as moving phase, reacted rear employing salt ion gradient elution (or low salt buffer wash-out) and obtained polyethyleneglycol modified albumen.
In preceding method, the filler of described hydrophobic chromatography chromatographic column comprises HiTrap Butyl FF, HiTrap Octyl FF or HiTrap Phenyl FF etc.The hydrophobic chromatography chromatographic column adopted in the present invention is prepackage drainage column (HiTrap Butyl FF, HiTrap Octyl FF and HiTrap Phenyl FF) that can commercially available acquisition, and column volume is 1mL.
In preceding method, described polyethyleneglycol modified dose comprises polyoxyethylene glycol-butyraldehyde, polyoxyethylene glycol-propionic aldehyde or polyethanediol succinate etc., and its molecular size range is 5kDa-40kDa, preferably 20kDa.
In preceding method, described high salt state refers to albumen or polyethyleneglycol modified dose is dissolved in the phosphate buffered saline buffer (as the PB damping fluid of 20mM pH6.0) that contains 2-3M sodium-chlor.
Adopt aforesaid method to prepare the single better and high PEG modified protein of activity keeping rate, the single modification rate of gained PEG modified protein reaches more than 40%, and the activity keeping rate reaches more than 80%, for the PEG that realizes albumen modifies, provides novel process.
The accompanying drawing explanation
The wash-out collection of illustrative plates that Fig. 1 is the N,O-Diacetylmuramidase solid phase PEG modified outcome based on the hydrophobic chromatography chromatographic column in the embodiment of the present invention 1.
Fig. 2 carries out the result of SDS-PAGE gel electrophoresis analysis to N,O-Diacetylmuramidase drainage column solid phase PEG modified outcome in the embodiment of the present invention 1; Wherein, a is the wild-type N,O-Diacetylmuramidase, the elution peak that b is N,O-Diacetylmuramidase drainage column solid phase PEG modified outcome; M is the standard protein molecular weight.
Fig. 3 is the result that adopts CM ionic chromatography post to carry out separation and purification to N,O-Diacetylmuramidase drainage column solid phase PEG modified outcome in the embodiment of the present invention 1.
The SDS-PAGE gel electrophoresis analysis result that Fig. 4 is each elution peak in Fig. 3; Wherein, m is the standard protein molecular weight, and a is the wild-type N,O-Diacetylmuramidase, and b is that N,O-Diacetylmuramidase drainage column PEG modifies products therefrom, the elution peak 1 and 2 in c, d difference corresponding diagram 3.
The wash-out collection of illustrative plates that Fig. 5 is the rhaFGF solid phase PEG modified outcome based on the hydrophobic chromatography chromatographic column in the embodiment of the present invention 5.
The SDS-PAGE gel analysis result that Fig. 6 is each elution peak in Fig. 5; Wherein, contrast as wild rhaFGF elution peak a, b, c in a, b, c difference corresponding diagram 5.
Fig. 7 modifies the circular dichroism spectrum analytical results of gained PEG-rhaFGF and wild rhaFGF based on hydrophobic chromatography chromatographic column solid phase PEG in the embodiment of the present invention 10.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The N,O-Diacetylmuramidase solid phase PEG of embodiment 1 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 2M NaCl, pH6.0,2MNaCl) HiTrap Butyl FF chromatographic column (column volume CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,2M NaCl) N,O-Diacetylmuramidase being mixed with to concentration is the 1mg/mL lysozyme soln, getting 5mL take flow velocity and is splined on HiTrap Butyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse to passing without albumen.
(3) to contain 30mM NaBH
3PB damping fluid (the 20mM of CN, pH6.0,2MNaCl) 2mg/mL polyoxyethylene glycol-butyraldehyde (20kDa) solution of preparation 10mL, using this mPEG-butyraldehyde solution as moving phase, flow velocity with 1mL/min flows through HiTrap Butyl FF chromatographic column, column temperature maintains room temperature, HiTrap Butyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,2M NaCl) and continue to rinse HiTrap Butyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Butyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak (Fig. 1).N,O-Diacetylmuramidase drainage column solid phase PEG modified outcome is carried out to the SDS-PAGE gel electrophoresis analysis, and result as shown in Figure 2.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect modified mixture elution peak (Fig. 3).Each elution peak is carried out to the SDS-PAGE gel electrophoresis analysis, and result as shown in Figure 4.
(7) elution peak after desalination is splined on to CM-Sepharose(CV=1.0mL) the cation-exchange chromatography post, flow rate gradient cationic exchange coloum with the PB damping fluid that contains 0-2M NaCl with 1mL/min, collect the PB buffer solution elution peak that wherein contains 0.3M NaCl, obtain the N,O-Diacetylmuramidase that PEG modifies.The PEG modification rate reaches 59%, and the activity keeping rate is 80%.
The N,O-Diacetylmuramidase solid phase PEG of embodiment 2 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 2M NaCl, pH6.0,2MNaCl) HiTrap Phenyl FF chromatographic column (column volume CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,2M NaCl) N,O-Diacetylmuramidase being mixed with to concentration is the 1mg/mL lysozyme soln, getting 5mL take flow velocity and is splined on HiTrap Phenyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) to contain 30mM NaBH
3PB damping fluid (the 20mM of CN, pH6.0,2MNaCl) 2mg/mL polyoxyethylene glycol-butyraldehyde (5kDa) solution of preparation 10mL, using this mPEG-butyraldehyde solution as moving phase, flow velocity with 1mL/min flows through HiTrap Phenyl FF chromatographic column, column temperature maintains room temperature, HiTrap Phenyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,2M NaCl) and continue to rinse HiTrap Phenyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Phenyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect the modified mixture elution peak.
(7) elution peak after desalination is splined on to CM-Sepharose(CV=1.0mL) the cation-exchange chromatography post, flow rate gradient cationic exchange coloum with the PB damping fluid that contains 0-2M NaCl with 1mL/min, collect the PB buffer solution elution peak that wherein contains 0.3M NaCl, obtain the N,O-Diacetylmuramidase that PEG modifies.The PEG modification rate reaches 61%, and the activity keeping rate is 80%.
The Chymetin solid phase PEG of embodiment 3 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 2M NaCl, pH6.0,2MNaCl) HiTrap Phenyl FF chromatographic column (column volume CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,2M NaCl) Chymetin being mixed with to concentration is the 1mg/mL protein solution, getting 5mL take flow velocity and is splined on HiTrap Phenyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) to contain 30mM NaBH
3PB damping fluid (the 20mM of CN, pH6.0,2MNaCl) 2mg/mL polyoxyethylene glycol-propionic aldehyde (20kDa) solution of preparation 10mL, using this solution as moving phase, flow velocity with 1mL/min flows through HiTrap Phenyl FF chromatographic column, column temperature maintains room temperature, HiTrap Phenyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,2M NaCl) and continue to rinse HiTrap Phenyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Phenyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect the modified mixture elution peak.
(7) elution peak after desalination is splined on to CM-Sepharose(CV=1.0mL) the cation-exchange chromatography post, flow rate gradient cationic exchange coloum with the PB damping fluid that contains 0-2M NaCl with 1mL/min, collect wherein each elution peak, obtain the Chymetin that PEG modifies.The PEG modification rate reaches 56%, and the activity keeping rate is 82%.
The Chymetin solid phase PEG of embodiment 4 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 2M NaCl, pH6.0,2MNaCl) HiTrap Butyl FF chromatographic column (column volume CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,2M NaCl) Chymetin being mixed with to concentration is the 1mg/mL protein solution, getting 5mL take flow velocity and is splined on HiTrap Butyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) to contain 30mM NaBH
3PB damping fluid (the 20mM of CN, pH6.0,2MNaCl) the 2mg/mL polyoxyethylene glycol of preparation 10mL-succinimide ester solution (25kDa), using this solution as moving phase, flow velocity with 1mL/min flows through HiTrap Butyl FF chromatographic column, column temperature maintains room temperature, HiTrap Butyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,2M NaCl) and continue to rinse HiTrap Butyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Butyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect the modified mixture elution peak.
(7) elution peak after desalination is splined on to CM-Sepharose(CV=1.0mL) the cation-exchange chromatography post, flow rate gradient cationic exchange coloum with the PB damping fluid that contains 0-2M NaCl with 1mL/min, collect each elution peak, obtain PEG and modify Chymetin.The PEG modification rate reaches 45%, and the activity keeping rate is 81%.
The rhaFGF solid phase PEG of embodiment 5 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 3M NaCl, pH6.0,3MNaCl) HiTrap Phenyl FF chromatographic column (CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,3M NaCl) polyoxyethylene glycol-butyraldehyde (40kDa) being mixed with to concentration is 2mg/mL, getting 10mL take flow velocity and is splined on HiTrap Phenyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) with the above-mentioned PB damping fluid (20mM that contains the 30mM sodium cyanoborohydride, pH6.0,3M NaCl) the 1mg/mL rhaFGF of preparation 5mL, using this protein solution as moving phase, flow velocity with 1mL/min flows through HiTrap Phenyl FF chromatographic column, column temperature maintains room temperature, HiTrap Phenyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,3M NaCl) and continue to rinse HiTrap Phenyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Phenyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak (Fig. 5).Each elution peak is carried out to the SDS-PAGE gel electrophoresis analysis, and result as shown in Figure 6.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect protein modified peak.The PEG modification rate reaches 42%, and the activity keeping rate is 92%.
The rhaFGF solid phase PEG of embodiment 6 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 3M NaCl, pH6.0,3MNaCl) HiTrap Butyl FF chromatographic column (CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,3M NaCl) polyoxyethylene glycol-butyraldehyde (20kDa) being mixed with to concentration is 2mg/mL, getting 10mL take flow velocity and is splined on HiTrap Butyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) with the above-mentioned PB damping fluid (20mM that contains the 30mM sodium cyanoborohydride, pH6.0,3M NaCl) the 1mg/mL rhaFGF of preparation 5mL, using this protein solution as moving phase, flow velocity with 1mL/min flows through HiTrap Butyl FF chromatographic column, column temperature maintains room temperature, HiTrap Butyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,3M NaCl) and continue to rinse HiTrap Butyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Butyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect protein modified peak.The PEG modification rate reaches 45%, and the activity keeping rate is 92%.
The rhbFGF solid phase PEG of embodiment 7 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 3M NaCl, pH6.0,3MNaCl) HiTrap Butyl FF chromatographic column (CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,3M NaCl) polyoxyethylene glycol-propionic aldehyde (20kDa) being mixed with to concentration is 2mg/mL, getting 10mL take flow velocity and is splined on HiTrap Butyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) with the above-mentioned PB damping fluid (20mM that contains the 30mM sodium cyanoborohydride, pH6.0,3M NaCl) the 1mg/mL rhbFGF of preparation 5mL, using this protein solution as moving phase, flow velocity with 1mL/min flows through HiTrap Butyl FF chromatographic column, column temperature maintains room temperature, HiTrap Butyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,3M NaCl) and continue to rinse HiTrap Butyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Butyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect protein modified peak.The PEG modification rate reaches 48%, and the activity keeping rate is 91%.
The rhbFGF solid phase PEG of embodiment 8 based on the hydrophobic chromatography chromatographic column modifies
Comprise the following steps:
(1) with the phosphate buffered saline buffer PB(20mM that contains 3M NaCl, pH6.0,3MNaCl) HiTrap Phenyl FF chromatographic column (CV=1mL) is carried out to pre-equilibration.
(2) adopt above-mentioned PB damping fluid (20mM, pH6.0,3M NaCl) polyoxyethylene glycol-propionic aldehyde (25kDa) being mixed with to concentration is 2mg/mL, getting 10mL take flow velocity and is splined on HiTrap Phenyl FF chromatographic column as 1mL/min, adopt PB damping fluid (20mM after completion of the sample, pH6.0,2M NaCl) rinse extremely without passing.
(3) with the above-mentioned PB damping fluid (20mM that contains the 30mM sodium cyanoborohydride, pH6.0,3M NaCl) the 1mg/mL rhbFGF of preparation 5mL, using this protein solution as moving phase, flow velocity with 1mL/min flows through HiTrap Phenyl FF chromatographic column, column temperature maintains room temperature, HiTrap Phenyl FF chromatographic column is wrapped up with lucifuge with masking foil simultaneously.
(4) moving phase is changed into to PB damping fluid (20mM, pH6.0,3M NaCl) and continue to rinse HiTrap Butyl FF chromatographic column, with flow velocity flushing 20 column volumes (CV) of 1mL/min.
(5) the flow velocity wash-out HiTrap Butyl FF chromatographic column with 1mL/min with salt-free PB damping fluid (20mM, pH6.0), collect elution peak.
(6) above-mentioned elution peak is splined on Sephadex G-25(CV=100mL) chromatography column, then use the flow velocity wash-out desalination of PB damping fluid (20mM, pH6.0) with 1mL/min, collect protein modified peak.The PEG modification rate reaches 51%, and the activity keeping rate is 92%.
The PEG of embodiment 9 based on the hydrophobic chromatography chromatographic column modifies the active testing of gained PEG-rhaFGF and wild rhaFGF
Comprise the following steps:
(1) the NIH3T3 cell in vegetative period of taking the logarithm, digest after centrifugal and be suspended in 1mL complete culture solution (89% low sugar DMEM substratum+10% foetal calf serum+1% penicillin/streptomycin), and it is 5 * 10 that counting is adjusted cell concn
3Individual cell/100 μ L/ holes are inoculated in 96 orifice plates and are placed in 37 ℃, 5%CO
2Cultivate 24h in incubator.
(2) discard the nutrient solution in 96 orifice plates, then wash once with PB, pat dry, add the hungry substratum of 100 μ L containing 0.4% foetal calf serum, the hungry 24h that processes.
(3) 4 experimental group are set, establish 2 multiple holes for every group, the rhaFGF standard substance that to give respectively final concentration be 50IU/mL, 0.1nM wild rhaFGF and solid state reaction gained PEG-rhaFGF(embodiment 6) carry out 2 times of gradient dilutions and (establish altogether 6 concentration, every hole adds the basic medium of 100 μ L containing medicine), cultivate 24h.
(4) the 96 every holes of orifice plate add 20 μ L MTT reagent (5mg/mL), in 37 ℃ of incubators, hatch 4h.Then discard liquid in hole, add 100 μ L DMSO and fully dissolve and mix, take 630nm as reference wavelength on microplate reader, in the 570nm place, read absorbance.Whole experiment repeats 4 times, and last disposal data carries out statistical analysis.Table 1 is to modify gained PEG-rhaFGF(embodiment 6 based on hydrophobic chromatography chromatographic column solid phase PEG) and the active testing result of wild rhaFGF.The result demonstration, drainage column solid phase PEG modifies the biologic activity that can retain preferably rhaFGF, and activity keeping, up to 92%, is modified the activity (61%) of gained PEG-rhaFGF far above traditional liquid phase P EG.Liquid phase P EG modifies with reference to Huang ZF, Lu MF, Zhu GH, et al, Acceleration of diabetic-wound healing with PEGylated rhaFGF in healing-impaired streptozocin diabetic rats.Wound Repair and Regeneration, 2011Sep-Oct, 19 (5): 633-644.
The biologic activity that the wild rhaFGF of table 1 and solid phase PEG modify gained PEG-rhaFGF compares
The circular dichroism spectrum analysis that the PEG of embodiment 10 based on the hydrophobic chromatography chromatographic column modifies gained PEG-rhaFGF and wild-type rhaFGF
The circular dichroism spectrum of albumen before and after adopting Jasco J715 circular dichroism spectrometer to measure to modify, sweep limit is 190-260nm(far-ultraviolet spectrum district), wild-type rhaFGF and PEG-rhaFGF(embodiment 6) protein concentration is 0.3mg/mL.Each sample is averaged after all scanning 6 times, and to scanning wavelength, mapping means molar ellipticity for net result [θ].Result shows (Fig. 7), and the secondary structure of drainage column modification gained PEG-rhaFGF and wild rhaFGF is in full accord.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the pegylation method of protein, is characterized in that, it is to take the pegylation method that the hydrophobic chromatography chromatographic column is the protein of solid state reaction carrier.
2. method according to claim 1, it is characterized in that, the strong albumen for hydrophobicity, under high salt state by protein adsorption on the hydrophobic chromatography chromatographic column, take polyethyleneglycol modified dose as moving phase, reacted rear employing salt ion gradient elution and obtained polyethyleneglycol modified albumen.
3. method according to claim 1, it is characterized in that, for the albumen a little less than hydrophobicity, under high salt state, polyethyleneglycol modified dose is adsorbed on the hydrophobic chromatography chromatographic column, take protein solution as moving phase, reacted rear employing salt ion gradient elution and obtained polyethyleneglycol modified albumen.
4. according to the described method of claim 1-3 any one, it is characterized in that, the filler of described hydrophobic chromatography chromatographic column comprises HiTrap Butyl FF, HiTrap Octyl FF or HiTrap Phenyl FF.
5. according to the method in claim 2 or 3, it is characterized in that, described polyethyleneglycol modified dose comprises polyoxyethylene glycol-butyraldehyde, polyoxyethylene glycol-propionic aldehyde or polyethanediol succinate.
6. method according to claim 5, is characterized in that, the molecular size range of described polyethyleneglycol modified dose is 5kDa-40kDa.
7. method according to claim 6, is characterized in that, the molecular size range of described polyethyleneglycol modified dose is 20kDa.
8. method according to claim 2, is characterized in that, the albumen that described hydrophobicity is strong comprises N,O-Diacetylmuramidase or Chymetin.
9. method according to claim 3, is characterized in that, the weak albumen of described hydrophobicity comprises recombinant acid fibroblast growth factor or recombination basic fibroblast growth factor.
10. according to the described method of claim 1-3 any one, it is characterized in that, described high salt state refers to albumen or polyethyleneglycol modified dose is dissolved in the phosphate buffered saline buffer that contains 2-3M sodium-chlor.
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| CN107177986A (en) * | 2017-05-24 | 2017-09-19 | 无锡协新毛纺织股份有限公司 | A kind of method for carrying out feld proofing to wool fabric using modified protein enzyme |
| CN108267517A (en) * | 2016-12-31 | 2018-07-10 | 江苏众红生物工程创药研究院有限公司 | A kind of method for detecting polyethylene glycol modified protein pharmaceutical purity |
| CN108634089A (en) * | 2018-01-11 | 2018-10-12 | 合肥工业大学 | A kind of preparation method of the high gelation bean protein powder of low sensitization |
| CN111484551A (en) * | 2020-03-19 | 2020-08-04 | 北京翼方生物科技有限责任公司 | Polyethylene glycol modified recombinant human alkaline fibroblast growth factor |
| CN113444139A (en) * | 2020-03-25 | 2021-09-28 | 中国科学院大学 | Synthesis and enzymatic hydrolysis promotion of pegylated D-amino acid-containing polypeptides |
| CN114853872A (en) * | 2022-04-27 | 2022-08-05 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
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Cited By (10)
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| CN106872624A (en) * | 2015-12-11 | 2017-06-20 | 江苏众红生物工程创药研究院有限公司 | A kind of method suitable for pegylated protein purity detecting |
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| CN108267517A (en) * | 2016-12-31 | 2018-07-10 | 江苏众红生物工程创药研究院有限公司 | A kind of method for detecting polyethylene glycol modified protein pharmaceutical purity |
| CN107177986A (en) * | 2017-05-24 | 2017-09-19 | 无锡协新毛纺织股份有限公司 | A kind of method for carrying out feld proofing to wool fabric using modified protein enzyme |
| CN108634089A (en) * | 2018-01-11 | 2018-10-12 | 合肥工业大学 | A kind of preparation method of the high gelation bean protein powder of low sensitization |
| CN111484551A (en) * | 2020-03-19 | 2020-08-04 | 北京翼方生物科技有限责任公司 | Polyethylene glycol modified recombinant human alkaline fibroblast growth factor |
| CN111484551B (en) * | 2020-03-19 | 2022-02-11 | 北京翼方生物科技有限责任公司 | Polyethylene glycol modified recombinant human alkaline fibroblast growth factor |
| CN113444139A (en) * | 2020-03-25 | 2021-09-28 | 中国科学院大学 | Synthesis and enzymatic hydrolysis promotion of pegylated D-amino acid-containing polypeptides |
| CN114853872A (en) * | 2022-04-27 | 2022-08-05 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
| CN114853872B (en) * | 2022-04-27 | 2023-11-17 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
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